P439: ASSOCIATING EX VIVO DRUG SENSITIVITY WITH METABOLIC STATUS IDENTIFIES EFFECTIVE COMBINATION STRATEGIES IN ACUTE MYELOID LEUKEMIA
Authors: D. A. Pereira-Martins, E. Griessinger, I. Weinhauser, J. L. Coelho-Silva, D. R. Silveira, L. Quek, A. Erdem, J. R. Hilberink, E. V. de Paula, S. T. Olalla Saad, E. Ammatuna, G. Huls, E. M. Rego, A. R. Lucena-Araujo, J. J. Schuringa
Journal: HemaSphere (2022)

Background: Metabolic reprogramming is a hallmark of cancer, and acute myeloid leukemia (AML) is no exception. Yet, we and others have shown that clear heterogeneity exists in metabolic programming between genetically distinct AML subtypes and altered function of mitochondria (mt) might be linked to chemoresistance in some patients. Aims: Here, we evaluated the mitochondrial function of AML cells in more detail. Methods: Retrospective analysis of the mtDNA content (mtDNAc) in a large cohort of Brazilian patients with de novo AML (03 centers, n=482, age:18-65y) treated with the 3 + 7 scheme, and sex- and age-paired healthy donors (HD, n=308), revealed higher mtDNAc in 34% of the AML samples when compared to the highest value of mtDNAc in the HD group. Results: High mtDNAc was independently associated with increased relapse risk. These findings were confirmed in a validation cohort of AML patients treated with the 3 + 7 scheme at the UMCG in the Netherlands (n=92, age:18-65y). Clustering analysis using label-free quantified proteome of sorted CD34+ cells from AML samples (n=30) associated high mtDNAc with oxidative phosphorylation (OxPhos) metabolism and L-GMP leukemia. Flow cytometry analysis for Hematopoietic progenitors (defined by CD34, CD38, CD123, and CD45RA gated inside the CD45+ cells) and mitochondrial markers (Mitotracker Green/DeepRed for mitochondrial mass/potential, respectively) in a set of primary AML samples (n=32), showed an enhanced proportion of L-GMP cells and a positive correlation with the mitochondrial potential (r=0.51) in AMLs with high mtDNAc. Consistently, oxygen consumption rate (OCR) and lactate production measured by Seahorse was increased in primary AML blasts (n=11) and AML cell lines (n=14) with high mtDNAc, suggesting increased energetic metabolism. Multiomics analysis of AML cell lines (n=19) revealed a positive correlation between mtDNAc and mitochondrial metabolism and apoptosis regulation. To evaluate the role of mtDNAc during drug-induced apoptosis, we treated primary AML blasts (n=67) with cytarabine (AraC, 100 nM) for 48h. We observed a negative correlation between mtDNAc and apoptosis rate (r=-0.75). Moreover, the reduction of mtDNAc in AML blasts (n=16) using shRNA targeting the POLG gene resulted in mixed phenotypes. POLG silencing impaired cell proliferation and induced monocytic differentiation for AMLs with high mtDNAc, while promoted cell proliferation in mtDNAclow AMLs, suggesting a completely distinct dependency on OxPhos for these 2 groups of AMLs. Pharmacological treatment of 9 AML cell lines with ETC complex I inhibitors (CIi; rotenone, 50 nM and metformin, 1 mM) in combination with AraC (10-500 nM) and Venetoclax (VEN, 10-1000 nM), resulted in a reduction of mtDNAc and functional respiration in AML cells. Combination therapy of CIi with AraC resulted in increased cell death in OxPhos - but not in glycolysis-driven AML cells. In contrast, the combination of CIi+VEN resulted in enhanced cell death in all AML cells, regardless of their metabolic state. Since metformin is an FDA-approved drug, we assessed the metformin-induced apoptosis, in combination with AraC or VEN in 64 primary AML samples ex vivo. Metformin treatment resulted in a reduction of basal OCR of AML blasts (n=7). The metformin-induced apoptosis rate positively correlated with mtDNAc (r=0.89), displaying additive effects with AraC- and VEN-induced apoptosis in AML samples. Summary/Conclusion: Overall, our findings suggest that mtDNA content can be used to identify patients with a higher risk of chemoresistance and supports the idea of repurposing metformin into AML therapy.